Method for preparing 213 Bi for therapeutic use

ABSTRACT

The present invention relates to the field of site directed therapy. More specifically it relates to site directed radio therapy. It provides a method for production of radioimmuno conjugates and an apparatus for radioimmuno therapy. The method, conjugates and apparatus can be practicalized without the need for radioactive shielding and/or airtight facilities. Without these restrictions the invention provides a simple and efficient means of therapy at the bed-side of the patient.

This is a continuation in part of U.S. Ser. No. 07/657,580, filed Feb. 19, 1991, now U.S. Pat. No. 5,246,691, issued Sep. 21, 1993, and a continuation of U.S. Ser. No. 08/097,471, filed Jul. 27, 1993, now abandoned.

The present invention relates to the field of site directed therapy. Nowadays there are a number of methods of site directed therapy which have been suggested to eliminate unwanted cells or infectious organisms from the body of a mammalian subject.

There are many fields of therapy in which said methods may be applied.

The most important ones seem to be immune diseases (either auto immune diseases or acquired immune diseases), cancer and viral or microbial infections.

BACKGROUND OF THE INVENTION

Site directed therapy is a method whereby a cytotoxic compound is delivered to the immediate vicinity of the target cell or infectious organism. This is usually done by coupling a targeting moiety to the cytotoxic compound.

This targeting moiety recognizes a structure in, on, or near the target. Known targeting moieties include, but are not limited to, antibodies, more specifically monoclonal antibodies and more preferably human monoclonal antibodies, nucleic acids, receptor directed ligands and the like.

Cytotoxic compounds can be for instance drugs, such as adriamycin, toxins such as ricin A and radioisotopes.

Radioisotopes cannot only be used for therapy, but they can also be used to identify the site or sites of the target (imaging). This invention provides methods of therapy and imaging using a conjugate of a targeting moiety and at least one radioisotope.

Therapy with targeting moieties is widely known. Targeting can be accomplished by aiming the targeting moiety directly to the wanted site, but it may also be directed to another targeting moiety which is directed to the wanted site (so called pretargeting). Pretargeting offers an advantage over direct targeting when the specificity of the targeting moieties is not sufficient. By using a first localizing moiety followed by a second one coupled to a cytotoxic compound, the amount of cytotoxic compound delivered to non-target sites can be lowered significantly.

Known targeting moieties, such as antibodies, often cannot be provided with a large amount of cytotoxic compounds without hampering their targeting specificity.

Therefore it has often been suggested to use a carrier molecule, such as HSA or a nucleic acid, or a polymer, which can be loaded with a high number of cytotoxic compounds and coupled to a targeting moiety.

All of the above-mentioned variations on the theme of site directed therapy and/or imaging can be used more advantageously with the present invention.

A well established problem in the field of imaging and site directed radiotherapy is to find a suitable radioisotope. Apart from the amount of energy that is released upon their decay, which should be sufficient to be measurable outside the subject in the case of imaging and sufficiently lethal to the target in the case of therapy, there is also a problem in finding an isotope with a suitable half-life.

An isotope with a long half life cannot be chosen because of the biological half life of the targeting moiety, which means that most of the isotopes will decay after disintegration of the conjugate. This decay after the disintegration of the conjugate will lead to cytotoxicity to other cells or tissues than the target.

Furthermore, all conjugates which do not localize will be secreted from the body and present a radio active waste problem.

It is also not practical to choose a radioisotope with too short a half life, because of packing and shipping delays and because the institution carrying out the therapy must be equipped to make the conjugate, transport it to the patient and administer it in a very short interval of time, otherwise most of the radioisotope will have decayed before entering the body, let alone localization at the target site.

The isotopes used for imaging usually are gamma emitting isotopes, for therapy auger electron emitting α,β-emitting, or α-emitting isotopes may be used.

Most preferred for the present invention are α-emitting isotopes.

The short-range cell-killing effect of α-particles is enormous: a 1 mm diameter tumor, comprising maybe 600,000 cancer cells needs about 6 α-particles of 6 MeV per cell to deliver a dose of 600 rad, causing a 99.9% cell-kill ratio, and that specifically because of the stochastic nature of the hit- and kill-mechanism.

However, due to the same stochastic nature, a 10 times lower α-radiaton dose will enhance the cell-survival ratio with a factor 500: more than 50% of the cells (or non-tumor cells in similar morphology for that matter) would survive a 60 rad α-radiation dose, equivalent to 0.6 α-particles per cell.

This characteristic would make an effective α-radioimmunotherapy within reach, provided that a "quality factor" for the isotope-antibody conjugate of 10 or better can be obained. It is the purpose of the present invention to contribute towards this goal in a most essential manner.

The quality factor is a ratio between localized antibody at the target site, divided by the antibody "sticking" to other tissue.

The notion of using α-particles emanating radioisotopes as agents for the killing of tumor cells was already mentioned in the literature during the mid-fifties. Since then other potential candidate-isotopes were and are being proposed, of which a good summary is given by Fisher (1) and by Wilbur (2) which brings the list to (with their half-lifes between parentheses): ²²³ Ra (11.4 d), ²²⁵ Ac (10 d), ²²⁴ Ra (3.6 d), ²²⁵ Fm (20 h), ²¹¹ At (7,2 h), ²¹² Bi (60 m), and ²¹³ Bi (47 m).

Although important publications appear regularly in the literature regarding microdosimetry, antibody-isotope coupling techniques, pre-clinical in vitro and in vivo experiments, no clearly defined, larger scale clinical experiments are being done until now, for a variety of reasons:

a. no human monoclonal antibodies with proven sufficient quality are available yet,

b. no biological safety data are available for antibody coupling agent (the latter for the binding of the radioisotope) combinations,

c. some isotopes may not become available for large scale application at acceptable costprices (²²⁵ Fm),

d. isotopes may be too difficult and therefore too expensive to obtain because of the necessary procurement process (²¹¹ At from ²⁰⁹ Bi by a (α, 2n) reaction in a cyclotron and subsequent isolation plus purification),

e. other isotopes do have a Rn-isotope as first daughter in their decay sequence, allowing redistribution of daughter nuclei before decay (²²⁴ Ra, ²²³ Ra), and also necessitating gas-tight reaction conditions,

f. some isotopes may have a relatively long living daughter isotope somewhere in their decay sequence (²²⁴ Ra, ²²³ Ra, ²²⁵ Ac), also with the chance that daughters thereof may redistribute before decay,

g. the radioactive halflife of some isotopes is so long that most of the activity leaves the patient undecayed, resulting in a waste problem (²²³ Ra, ²²⁴ Ra, ²²⁵ Ac), or

h. the halflife of the isotope is so short that most of the isotope decays before reaching its ultimate target (²¹² Bi, ²¹³ Bi),

i. sufficient precursor material may not be available to extract at one time the necessary amount of isotope for a single patient treatment (²¹² Bi, ²¹³ Bi), and,

j. isotopes producing hard gamma rays in their isotope decay need shielding facilities to prevent radiation hazards to technicians and nursing personnel.

One or more of the arguments listed above will make it very difficult, if not impossible for some of the isotopes to ever be used on a large scale for α-radio-immunotherapy, and that in particular if one or more of the others can be used on acceptable technical, logistical and financial conditions.

The closest prior art to the present invention (the French patent application FR-A-2 527 928) discloses a method to produce conjugates of an antibody and ²¹² Bi. However, these conjugates still suffer from the drawbacks mentioned above under e, h, i and j.

SUMMARY OF THE INVENTION

The present invention provides a method for producing a conjugate of a targeting moiety and at least one radioisotope at or near the bedside of a patient, characterized in that a relatively long lived radioisotope of which the daughters in the decay sequence predominantly emit α and/or β rays, is loaded on an appropriate medium out of which medium a relatively short lived isotope is eluted and coupled to a targeting moiety.

Relatively long lived in this context means that the radioisotopes have a decay time in the order of several days, which enables sufficient time for packing and shipping. Relatively short lived in this context mean that the radioisotopes have a decay time in the order of minutes or hours.

With a decay sequence in which predominantly α- and/or β-rays are emitted is meant a decay sequence which does not cause danger for radiation hazards caused by gamma rays to people working with the compounds without the burden of applying protective shielding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an apparatus according to the invention.

An eluent containing a targeting moiety passes from a vessel (1) through an ion exchange column (3) containing a bound parent radiometal, wherein a daughter radioisotope binds to the targeting moiety. The element containing the conjugate is mixed with infusion liquid from vessel (2) at junction (6) and administered to the patient (4). Optionally there is additional length of tubing (5) between column (3) and junction (6) to correct for the half lives of intermediate daughter isotopes.

FIG. 2 depicts the decay chain of ²²⁵ Ac.

FIG. 3 depicts another apparatus according to the invention.

An eluent passes from a vessel (7) through an ion exchange column (3) where a daughter radioisotope is stripped from that column.

The eluent containing the isotope is mixed with a liquid from a vessel (1) containing a targeting moiety, so that the isotope becomes bound to the targeting moiety. The resulting fluid is mixed with infusion liquid from vessel (2) at junction (6) and administered to the patient (4). Optionally the eluent containing the isotope may be lead through an additional length of tubing (5) to correct for the half-life of intermediate daughter isotopes.

FIG. 4 shows the elution behavior of ²¹³ Bi at several HCl and HI concentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An important aspect of the present invention is that the radioconjugate can be made or quasi be made at the site of therapy. Due to the decay sequence which results mainly in α- and/or β-radiation it has become possible that no protection against radiation is necessary. This is extremely useful, because due to the absence of gamma radiation it has become possible that the conjugation can be done at or near the bed side without the necessity to apply radiation shielding or isolation of the patient. This is not only preferable from the point of radiation danger but is also gives advantages for the availability of the short-living isotope. This isotope can be prepared in the neighboorhood of the patient allowing for a rapid administration and prevention of loss of therapeutic action caused by the rapid decay of the isotope. In this way it has become possible to use short lived radioisotopes for therapy.

An ion exchange column or another appropriate substrate filled with the long lived isotope can be placed at or near the bedside, for instance, where the short-lived isotope can be eluted by washing the substrate with a suitable solution. After elution the short lived isotope is coupled to the targeting moiety and (optionally together with an infusion solution) the conjugate can be administered. This can all be done in a continuous mode with an apparatus according to the invention as shown in FIG. 1 or FIG. 3, or in an intermittent mode by using ordinary laboratory glassware.

Of course it may also be made possible to add the targeting moiety to the eluting solution so that the coupling takes place in the column.

This invention primarily addresses the use of the shortest-lived isotope from the list mentioned above, ²¹³ Bi. The invention enables the person skilled in the art to milk this isotope by a continuous or an intermittent extraction-method from one of its precursors, ²²⁵ Ac, at the bed-side of the patient, or in the nearestby hospital laboratory facility, to link the ²¹³ Bi in a continuous or an intermittent manner onto the targeting moiety, to either or not mix the conjugate solution with an infusion liquid and to administer this mixture intravenously to the patient--for example as is schematically pictured in FIG. 1.

At first sight this procedure might seem extremely wasteful, because ²²⁵ Ac, itself being an α-emitting isotope, produces three potentially therapeutically useful α-particles before yielding the ²¹³ Bi-isotope, as is shown in FIG. 2. However, the source material for ²²⁵ Ac, ²²⁹ Th, and thereby also the ²²⁵ Ac itself, can be made available at sufficiently low cost to allow it to be used in the proposed manner on economically justifiable terms.

The use of ²¹³ Bi is not only preferable from a viewpoint of radiation hazards. It is also preferable because no gaseous isotopes occur in the decay sequence of its precursors. This is advantageous over the use of other isotopes which have a decay with a gaseous isotope which necessitates the handling and reaction environment to be air-tight. Milking, conjugation and administration of ²¹³ Bi are not hampered by the necessity for having air-tight conditions and the reactions can be done under normal conditions.

The targeting moiety may preferably be a monoclonal antibody, or a fragment or a derivative thereof. Preferably such an antibody is a human or a humanized antibody to prevent immunologic reactions to the antibody. Non-human antibodies are mostly of murine origin. These, like all other foreign proteins, are highly immunogenic in man. The phenomenon of HAMA, human anti mouse antibodies, is well known in the field and severely limits the use of mouse derived antibodies in diagnostic and especially in therapeutic applications in human beings. A single application of a murine antibody is usually sufficient to mount an immune response that will prevent subsequent applications to be effective.

Of course fragments and/or derivatives of the targeting moieties can also be used, as long as they retain a substantial amount of target specificity. Thus, for this invention it should be understood that where a targeting moiety is mentioned one should also consider a fragment or a derivative thereof as part of the invention.

Preferably antibodies are directed against tumor associated antigens, such as CEA (Carcino-embryonic antigen), AFP (alpha-foetoprotein), FHAP (fast homoarginine-sensitive alkaline phosphatase), p97 (melanome specific), and EL-1 (elongation factor 1).

Another preferable targeting moeity is formed by a ligand for a cell surface receptor or a fragment or derivative of such a ligand. Examples of such ligands are agonists and/or antagonists of pharmacologically active receptors, but also T cell epitopes which can bind to the T cell receptor are prefered.

Another aspect of the invention provides a method for treating numerous patients with one ion exchange column loaded with isotope. The amount of isotope loaded depends on the number of patients to be treated. The wanted isotope can be eluted from the column intermittently, with suitable intervals depending on the half-lifes in the decay chain.

With related tumours or infectious organisms the same targeting moiety (or mix of targeting moieties) may be used for various patients. For unrelated diseases there must be a means for changing the targetting moiety preparation.

The coupling of the isotope to the targeting moiety can be done in any suitable way, as long as the targeting specificity of the targeting moiety is not hampered to a substantial amount.

Preferably the coupling will be done through one of the now many known chelating agents. As already disclosed, it may be advantageous to couple the isotopes to a carrier, such as HSA, which of course can also be done through chelating agents. The advantage of a carrier is that a large number of radioisotopes can be brought to the target cell. Since it is assumed that several α-particles are necessary for the destruction of one target cell an increase in the number of isotopes in the direct neighbourhood of the target cell is preferable.

The invention also provides a conjugate as produced by the method of the invention, as well as a pharmaceutical formulation comprising such a conjugate.

A method is provided for producing the conjugate of a targeting moiety and a radioisotope and administering it to the patient without delay or any necessary actions of the therapist.

Another aspect of the invention provides an apparatus for carrying out site directed therapy or imaging.

The simplest way to describe the method and apparatus, subject of this invention, with reference to FIG. 1, is as follows:

A capillary column contains, by means of example, twice the amount of precursor-²²⁵ Ac needed for a single patient dose of ²¹³ Bi. Example: in a case the patient dose corresponds with 30 mCi (equals 2.10⁻⁹ g) of ²¹³ Bi over a 10 day period, the capillary column (3) will contain 200 μCi of ²²⁵ Ac (equals 4.10⁻⁹ g).

The ²²⁵ Ac is present in a 3⁺ form on a suitable ionexchange substrate. Upon its (continuously occurring) decay it is stripped from the column by a certain overdose of the eluent in flask (1) containing the appropriate targetting moiety capable of binding the isotope. The binding part of the targetting moiety and other chemical equilibrium conditions of the eluent-ionexchange system are chosen such that the ²¹³ Bi, for all practical purposes, quantitatively binds to the targetting moiety. The immediate daughter of ²²⁵ Ac, ²²¹ Fr has a radioactive decay halflife of 4.8 minutes. It is this isotope which acts via the very short-lived ²¹⁷ At as the direct precursor of ¹¹³ Bi. In case the ²²¹ Fr is not retained by itself or in the ion exchange substrate, the delaying effect of the ²²¹ Fr-halflife causes the need of a certain period of time between the decay of ²²⁵ Ac at and its stripping from the capillary column and the binding of the ²¹³ Bi onto the targetting moieties. The optimum value for such a delay is somewhere between the halflife of the ²²¹ Fr and the halflife of the ²¹³ Bi isotopes.

This delay can be effected by the length of tubing between the capillary (3) and the patient (4), if necessary enhanced by an extra length of intermediate tubing, as indicated in FIG. 1 as (5). The infusion liquid from flask (2) enters the patient, it is mixed with the isotope-containing eluate from column (3), as indicated as junction (6) in FIG. 1.

In order to obtain optimal stripping and conjugation conditions in the capillary column (3), it may be that the composition of the eluent in flask (1) is not optimal (for example its pH-value) for administration to the patient. Presuming that the volume rate of infusion liquid is an order of magnitude higher than of the eluate liquid, this can easily be countered for by a compensating off-balance (buffered) pH-value of the infusion liquid.

It is also possible that the binding of the targeting moiety is hampered by the physico-chemical properties of the eluent. Therefore, an other embodiment of the invention is represented in FIG. 3 where an eluens is lead from a vessel (7) through an ion exchange column (3) so that a radioisotope is stripped from that column. The eluens containing the isotope is mixed with a liquid from a vessel (1) containing a targeting moiety, so that the isotope is bound to the targeting moiety. The resulting fluid is mixed with infusion liquid from vessel (2) at junction (6) and administered to the patient (4). Optionally the eluens containing the isotope may be lead through an additional length of tubing (5) to correct for the half-life of intermediate daughter isotopes.

What the invention enables in terms of the development and the clinical use of α-radioimmunotherapy, in this case using ²¹³ Bi as the active cell-killing agent is:

"single patient kits" in the form of precursor with a halflife that is logistically managable regarding:

minimization of active material loss by radioactive decay during operations like packaging, transport, etc.,

safety in transportation over long distances and in handling in hospitals,

applicability in practice on a large scale in many hospitals without need for special precautions, regarding:

the handling of the material and the application procedures regarding the treatment of patients, all without complicated monitoring equipment,

collection and handling facilities for (urinous) waste,

maximal (and in case of continous extraction, almost total) use of the ²¹³ Bi after it is generated from the precursor isotope,

maximum flexibility in dose administration by the possibility of changing treatment time, allowing for a minimum range of single patient kit precursor concentration standards.

All these aspects then pertain precisely to fields where the short-range α-particles are most suited for their potential therapeutic uses like:

micrometastases (of less than 1 mm diameter) of various cancers,

cellular cancers like leukemias and

also, certain kinds of very localized autoimmune diseases, all of which can essentially be directly addressed either by the blood-circulation system or locally without the need for slow diffusion processes of the antibody-ligand-isotope complexes through intercellular space in order to find their ultimate destination.

A special advantage of intermittent administration of the therapeutic radioconjugates is the advantage which occurs by dose fractionation. Statistically it is possible to calculate the dose needed to kill 99.9% of the tumor cells with a dose of radioconjugate: assuming that a leukemic (monocellular, blood and marrow bone) tumor load of 1 kg exisits, which is roughly equal to 10¹² cells, and that 10 α-particles are needed to kill a cell (6 MeV), then 10¹³ α-particles would be needed, which corresponds with 50 mCi ²¹³ Bi. Thus for a single dose, which would kill 99.9% of the tumor cells 50 mCi ²¹³ Bi would be needed. The "dose versus survival" relation for this cell morphology with 6 MeV α-particles can be derived from the formula D/D₀ =-ln S, in which S=survival fraction, D=dose administered and D₀ =reference dose for 37% survival. From this formula the following Table of values can be calculated:

                  TABLE 1                                                          ______________________________________                                         Dose versus kill ratio for tumor cells. The numbers are the                    number of α-particles necessary to kill the given % of tumor             cells.                                                                         In case A 600 rad are necessary to obtain a 99% kill ratio. In case            B 2000 rad is assumed necessary for the same effect.                           Cell kill in %   Case A  Case B                                                ______________________________________                                         0                0       0                                                     1                0.015   0.05                                                  10               0.15    0.5                                                   40               0.7     2                                                     50               1       3                                                     60               1.3     4                                                     70               1.5     5                                                     90               3       10                                                    99               6       20                                                    99.9             9       30                                                    99.99            12      40                                                    99.999           15      50                                                    ______________________________________                                    

From this Table the effects of an intermittent, dose fractioned, administration can be read:

The effect of cell survival of successive doses of 5 mCi ²¹³ Bi in case A is as follows:

the first dose of 5 mCi equals 1 α-particle per cell, which gives 50% survival, which means that 0.5·10¹² cells remain;

the second dose of 5 mCi equals 2 α-particles per cell, which gives 20% survival, which means that 0.1·10¹² cells remain;

the third dose of 5 mCi equals 10 α-particles per cell, which gives 0.1% survival, which means that 0.1·10⁹ cells remain;

the fourth dose of 5 mCi equals 10,000 α-particles per cell, which means a total kill.

Thus it can be shown that by intermittent dosing a total dose of 4 times 5=20 mCi ²¹³ Bi is sufficient to give a total kill of the tumor cells. For clarity the effects of intermediate tumor growth and maximization of the number of targeting moieties on the tumor cells have been omitted. Nevertheless, it is clear that by intermittent administration the total load of radioactive material can be kept smaller.

Even in case B, which has a more unfavourable dose versus survival rate, and advantageous effect is realized:

    1.sup.st dose→1α/cell→75% survival→0.75·10.sup.12 cells

    2.sup.nd dose→1.3α/cell→70% survival→0.50·10.sup.12 cells

    3.sup.rd dose→2α/cell→60% survival→0.30·10.sup.12 cells

    4.sup.th dose→3α/cell→50% survival→0.15·10.sup.12 cells

    5.sup.th dose→6α/cell→25% survival→0.04·10.sup.12 cells

    6.sup.th dose→25α/cell→0.3% survival→0.1·10.sup.9 cells

    7.sup.th dose→10,000α/cell→total kill after 35 mCi.

There are two ways presently known to obtain ²²⁹ Th as a precursor for the ²²⁵ Ac-source-isotope:

from stockpiled ²³³ U, by its natural α-decay. Batches of ²³³ U were made in nuclear breeder reactors about 30 years ago, but never used as nuclear fuel.

Some of the ²³³ U was separated from the bulk-²³³ Th, from which it was made, so that the now available ²²⁹ Th can be obtained in highly pure form.

by high neutron flux irradiation from natural ²²⁶ Ra, with ²²⁷ Ac as an intermediate product. Futher irradiation of this ²²⁷ Ac yields roughly equal amounts of ²²⁹ Th and ²²⁸ Th, the latter with much shorter halflife (2 years) than the ²²⁹ Th. On the one hand this complicates the extraction of ²²⁵ Ac considerably, but in properly equipped installations it may on the other hand yield ²²⁴ Ra, an α-emitter with a 3.7 day halflife. When the Ra is properly isolated, it may be used as a source for ²¹² pb.

The 10.5 hour halflife of ²¹² Pb will cause considerable complications in handling. However, when these are properly taken care of, one may envisage to use the ²¹² Pb-isotope in the same manner as the ²²⁵ Ac in this invention as a bed-side source of ²¹² Bi, which for al practicle purposes acts as an α-emitter with a halflife of 1.0 hour.

EXAMPLES Example 1

The separation chemistry of the various radioactive elements mentioned in the text before has been sorted out decades ago and is well-documented in the public literature. Examples are references (3) and (4). ²²⁵ Ac can be separated from ²²⁹ Th on a Dowex 50 ionexchanger by stripping with 4N HNO₃. After evaporation of the acid, the ²²⁵ Ac can be dissolved again in 0.5N HNO₃ in a fixed concentration and absorbed in the appropriate amount on Dowex 50, which then becomes the material in the mini-column (3) of FIG. 3.

Example 2

0.68±0.07 mCi of ²²⁵ Ac was obtained from the European Joint Research Centre. This was loaded on a MP-50 cation exchange resin (Bio-Rad). The formed ²¹³ Bi was eluted with a mixture of 50:50 10% NH₄ Ac:MeOH with a pH of 6.75. An autoburet was used to deliver 35 μl of eluant per minute; alternatively, manual elution was done at 50 μl amounts of eluant per minute.

In a few experiments it was necessary to purify the ²¹³ Bi. This was accomplished by heating the eluant to dryness in a 10 ml beaker containing 0.5 ml of conc. HNO₃. After evaporation under an IR lamp, the bismuth activity was transferred to a column of MP-50 resin (2×30 cm, pre-equilibrated with 0.1M HNO₃). The resin was washed with 0.2 ml H₂ O. Then the ²¹³ Bi was eluted with 0.5 ml of HCl and HI. Various concentrations of HCl and HI have been tried. FIG. 4 shows the elution patterns for ²¹³ Bi. In all cases the elution is rapid and quantitative. All of the isotope can be obtained within 5 to 10 minutes after the start of the elution.

Example 3

Radiolabeling was done by adding enough 3M NH₄ Ac to the ²¹³ Bi stock to achieve pH 4.0-5.0. Then 53 μl or 106 μl of a 4.7 mg/ml solution of monoclonal antibody B3 coupled with the chelator CHX-DTPA (cyclohexyldiethylenetriaminepenta acetic acid) according to the method described in (5) were gently mixed into the solution. After a fifteen minute reaction time, 1.5 μl of 0.1M EDTA were added. The solution was transferred to a 1 ml syringe with 0.2 ml wash. The solution was then injected into the HPLC (high pressure liquid chromatography) having a TSK 3000 column. The buffer was 0.02M MES/Cl⁻ (MES=morpholino ethane sulfonic acid), 0.15M NaCl, pH 6.5. Elution of the B3 antibody occurred at 7.5 minutes. The amount of ²¹³ Bi incorporated into the antibody was monitored with an in-line radiochemical detector (Beckman). All activity measurements of ²¹³ Bi were corrected for decay (t_(1/2) =45.6 min). Results are depicted in Table 2. Activities of ²²⁵ Ac, ²²¹ Fr or ²¹⁷ At were not detectable in any of the ²¹³ Bi elution products.

                  TABLE 2                                                          ______________________________________                                         Results of radiolabeling experiments incorporating                             .sup.213 Bi into mAb B3-CHX-DTPA.                                                                           .sup.213 Bi                                       Acid    Vol. acid μl                                                                           μg mAb recovered in mAb (%)                              ______________________________________                                         2M HCl  210        250       43.4 (37%)                                        2M HCl  210        500       45.0 (25%)                                        0.1M HCl                                                                               500        500       3.4 (7%)                                          0.1M HI                                                                        ______________________________________                                    

Example 4

In the same way as described in Example 2 and 3, ²¹³ Bi was eluted from ²²⁵ Ac and coupled to a targeting moiety. For this experiment a conjugate of monoclonal antibody M195 and the chelator CHX-DTPA was used. Table 3 summarizes the results.

                  TABLE 3                                                          ______________________________________                                         Results of radiolabeling experiments incorporating                             .sup.213 Bi into mAb M195-CHX-DTPA.                                            μg antibody                                                                               .sup.213 Bi recovered (%)                                        ______________________________________                                         50            198 (53%)                                                        25            107 (39%)                                                        ______________________________________                                    

REFERENCES

(1) D. R. Fisher: "α-particle emmitters in medicine", proceedings of a symposium held at Loews L'Enfant Plaze Hotel, Washington, D.C., September 21 and 22, 1989, pages 194-214, published by the American College of nuclear physicians.

(2) D. S. Wilbur: "Potential use of α-emitting radionuclides in the treatment of cancer", Antibody, Immunoconjugates, and Radiopharmaceuticals, volume 4, number 1, 1991, pages 85-97, published by Mary Ann Liebert, Inc.

(3) T. Mitsugashira: "Preparation of traces for actinium, thorium, protactinium and uranium", SPEY, Min. Educ. Sci. & Cult., Tokyo, 9, 1984, pages 111-116.

(4) S. Suzuki: "Solution chemistry of light actinide elements", Japan-US seminar on Thorium fuel reactors--proceedings, Nara, Japan, 18-22 October 1982 (Tokyo, 1985) pages 137-143.

(5) Mirzadeh, S., Brechbiel, M. W., Atcher, R. W., Gansow, O. A., Bioconjugate Chem., volume 1, 1990, 59-65. 

I claim:
 1. A method for producing a conjugate of a targeting moiety and ²¹³ Bi near the bedside of a patient, comprising the following steps in the order as stated: loading ²²⁵ Ac onto an appropriate binding medium for binding the ²²⁵ Ac, eluting from the medium ²¹³ Bi and coupling the eluted ²¹³ Bi free of ²²⁵ Ac to a targeting moiety to form a conjugate, wherein the targeting moiety is selected from the group consisting of a monoclonal antibody and an antigen binding fragment of an antibody, wherein the targeting moiety has specificity for a tumor associated antigen.
 2. A method for α particle immunotherapy comprising administering an amount of ²¹⁵ Bi effective for killing target cells in a mammal, wherein said target cells are in micrometastases having a diameter of about 1 mm or less or are in cellular diseases, said method comprising the following steps in the order as stated: providing a sufficient quantity of ²²⁵ Ac to produce the effective amount of ²¹³ Bi through radioactive decay; binding the ²²⁵ Ac onto a binding medium for immobilizing ²²⁵ Ac; eluting ²¹³ Bi produced by bound ²²⁵ Ac from the binding medium; coupling the eluted ²¹³ Bi free of ²²⁵ Ac to a targeting moiety to form a conjugate, said targeting moiety selected from the group consisting of an antibody and an antigen binding fragment of an antibody and having specificity for an antigen found on said target cells; administering the conjugate to the mammal; and permitting the conjugate to contact target cells in the mammal.
 3. The method of claim 2, wherein the conjugate is administered intermittently in fractions of the total amount of conjugate required to administer an effective amount of ²¹³ Bi for killing all target cells in the mammal, and wherein a sufficient number of fractions of sufficient quantities of conjugate are administered to kill essentially all target cells contacted, the total quantity of α radiation administered to the mammal being less than the total quantity necessary to kill essentially all target cells by administering a single dose of conjugate. 